Recently, a novel archaeal phylum, the Nanoarchaeota, was identified. The representative species Nanoarchaeum equitans is an extremely tiny (nano-sized), hyperthermophilic anaerobe which was isolated from a submarine hot vent at the Kolbeinsey ridge, north of Iceland (see, Huber, H. et al., 2002, Nature 417, 63-67). This organism grows on the surface of a specific crenarchaeal host, Ignicoccus sp. strain KIN4/I, under strictly anaerobic conditions between 70 and 98° C. The genome (490,885 base pairs (hereinafter, referred to as “bp”)) of N. equitans, one of the smallest microbial genomes, has been completely sequenced (see, Waters, E. et al., 2003, Proc. Natl. Acad. Sci. USA 100, 12984-12988). It was deduced from the genome sequence analysis that N. equitans is a parasite for Ignicoccus sp. strain KIN4/I.
Deoxyribonucleic acid polymerases (DNA polymerases; E.C. number 2.7.7.7) are enzymes that synthesize DNA in the 5′ to 3′ direction on template DNA. These enzymes play leading roles in cellular DNA replication and repair (see, Lehninger, A. L. et al., 1993, Principles of Biochemistry, 2nd ed., Worth Publishers). Beginning with the discovery and characterization of DNA polymerase I from Escherichia coli by Kornberg and colleagues in 1957 (see, Kornberg, A. & Baker, T., 1992, DNA Replication, 2nd ed., Freeman Company), a variety of DNA polymerases have been isolated and identified from prokaryotic and eukaryotic sources. These DNA polymerases have been classified into five major groups based on amino acid sequence similarity: families A, B, C and D, which include DNA polymerases having high similarity to E. coli DNA polymerase I, II, III a subunit, and Pyrococcus furiosus DNA polymerase II, respectively; and family X, which includes other DNA polymerases not belonging to the A to D families (see, Braithwaite, D. K. & Ito, J., 1993, Nucleic Acids Res. 21, 787-802; Cann, I. K. O. & Ishino, Y., 1999, Genetics 152, 1249-1267).
Thermostable DNA polymerase was initially isolated and identified from a thermophile, Thermus aquaticus YT-1, by Chien et al. in 1976 (see, Chien, A. et al., 1976, J. Bacteriol. 127, 1550-1557). Thereafter, studies were made with some thermophiles, but these did not attract particular interest. However, with the development of a PCR technique using thermostable DNA polymerase by Saiki et al. in 1988 (see, Saiki, R. K. et al., 1988, Science 239, 487-491), thermostable DNA polymerases became of great interest, and these enzymes have been competitively developed from several thermophiles and hyperthermophiles. In particular, thermostable DNA polymerases from hyperthermophilic archaeons, such as Thermococcus litoralis and P. furiosus, have been used in PCR requiring high fidelity because they have 3′→5′ exonuclease activity (this activity is known as proofreading activity) along with DNA polymerization activity (see, Mattila, P. et al., 1991, Nucleic Acids Res. 19, 4967-4973; Lundberg, K. S. et al., 1991, Gene 108, 1-6).
Inteins are protein insertion sequences that are embedded in-frame within precursor protein sequences. These sequences are removed from the precursor protein by a self-splicing process, and thus do not affect the structure and activity of the final protein made from the precursor protein (see, Perler, F. B. et al., 1994, Nucleic Acids Res. 22, 1125-1127). Protein splicing is a post-translational processing event in which the intein is precisely self-excised from a precursor protein with concomitant ligation of the flanking protein sequences, exteins, by a normal peptide bond (see, Kane, P. M. et al., 1990, Science 250, 651-657). Naturally occurring inteins, which are present in proteins of organisms, can be grouped into three types according to their structural organization: inteins, which have both self-splicing and homing endonuclease domains; mini-inteins, which lack the endonuclease domain and have the splicing domain; and split mini-inteins, lacking the endonuclease domain, in which the splicing domain exists as a split form on two separate genes, and are therefore spliced in trans (see, Martin, D. D. et al., 2001, Biochemistry 40, 1393-1402).
Neq DNA polymerase is encoded by two genes, which are separated by 83,295 bp on the chromosome and individually contain a deduced split mini-intein sequence (see, Waters, E. et al., 2003, Proc. Natl. Acad. Sci. USA 100, 12984-12988). The sequences of naturally occurring split mini-inteins, among about 180 known inteins, have been found only in several cyanobacterial C-type DNA polymerase III α subunits (hereinafter, referred to as “DnaE proteins”) (see, Caspi, J. et al., 2003, Mol. Microbiol. 50, 1569-1577). Among them, various studies have been made only on Synechocystis sp. PCC6803 DnaE protein (hereinafter, referred to as “Ssp DnaE protein”)(see, Wu, H. et al., 1998, Proc. Natl. Acad. Sci. USA 95, 9226-9231; Evans, T. C., Jr. et al., 2000, J. Biol. Chem. 275, 9091-9094; Martin, D. D. et al., 2001, Biochemistry 40, 1393-1402). This protein is different from Neq DNA polymerase in that it is derived not from archaea but from bacteria, is not a thermostable protein but a mesophilic or psychrophilic protein, and is not a B-type DNA polymerase but a C-type DNA polymerase. In addition, Methanothermobacter thermautotrophicus B-type DNA polymerase is encoded by two separated genes, but polypeptides made therefrom lack an intein and are thus active as a dimmer (see, Kelman, Z. et al., 1999, J. Biol. Chem. 274, 28751-28761).
PCR is a technique for exponentially amplifying a trace amount of template DNA using a DNA polymerase and primers. PCR amplification occurs in repeated cycles of three steps: DNA denaturation at 94° C., primer annealing at 40-65° C. and DNA extension at 72° C. Since the reaction requires high temperature, it is indispensably necessary to develop thermostable DNA polymerases, which are the most important factor in PCR, for the development and application of various PCR techniques (see, Erlich, H. A., 1989, PCR Technology: Principles and Applications for DNA Amplification, Stockton Press). Thermostable DNA polymerases are enzymes that are very useful in the identification and amplification of genes, DNA sequencing and clinical diagnosis by PCR. These enzymes are used in a wide spectrum of fields ranging from genetic engineering and molecular biology experiments to the diagnosis of hereditary diseases, early diagnosis of oncogenes and viral genes, and forensic medicine, and are thus increasing in demand.
To date, there has been no report involving the expression of any protein-encoding gene and the purification, biochemical properties and industrial application of any protein from the hyperthermophilic nanoarchaeon N. equitans. Also, there has been no report stating that an archaeal protein, a thermostable protein and a B-type DNA polymerase possess a split mini-intein.
Based on this background, the present inventors performed the sequence analyses of two genes encoding B-type DNA polymerase from N. equitans, and established methods of preparing active Neq DNA polymerase using a genetic engineering technique. The present inventors found that the active Neq DNA polymerase prepared according to the methods is applicable to general PCR and to PCR in the presence of deoxyuridine 5′-triphosphate (hereinafter, referred to as “dUTP”), thereby leading to the present invention.